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Voltage inactivation of Ca²⁺ entry and secretion associated with N- and P/Q-type but not L-type Ca²⁺ channels of bovine chromaffin cells

Mercedes Villarroya *, Román Olivares *, Ana Ruíz *, María F. Cano-Abad *, Ricardo de Pascual *, Richard B. Lomax *, Manuela G. López *, Inés Mayorgas *, Luis Gandía * and Antonio G. García *¹

MS 8592 Received 4 August 1998; accepted after revision 12 January 1999.

	ABSTRACT

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1. In this study we pose the question of why the bovine adrenal medullary chromaffin cell needs various subtypes (L, N, P, Q) of the neuronal high-voltage activated Ca²⁺ channels to control a given physiological function, i.e. the exocytotic release of catecholamines. One plausible hypothesis is that Ca²⁺ channel subtypes undergo different patterns of inactivation during cell depolarization.

2. The net Ca²⁺ uptake (measured using ⁴⁵Ca²⁺) into hyperpolarized cells (bathed in a nominally Ca²⁺-free solution containing 1·2 mM K⁺) after application of a Ca²⁺ pulse (5 s exposure to 100 mM K⁺ and 2 mM Ca²⁺), amounted to 0·65 ± 0·02 fmol cell^-1; in depolarized cells (bathed in nominally Ca²⁺-free solution containing 100 mM K⁺) the net Ca²⁺ uptake was 0·16 ± 0·01 fmol cell^-1.

3. This was paralleled by a dramatic reduction of the increase in the cytosolic Ca²⁺ concentration, [Ca²⁺]_i, caused by Ca²⁺ pulses applied to fura-2-loaded single cells, from 1181 ± 104 nM in hyperpolarized cells to 115 ± 9 nM in depolarized cells.

4. A similar decrease was observed when studying catecholamine release. Secretion was decreased when K⁺ concentration was increased from 1·2 to 100 mM; the Ca²⁺ pulse caused, when comparing the extreme conditions, the secretion of 807 ± 35 nA of catecholamines in hyperpolarized cells and 220 ± 19 nA in depolarized cells.

5. The inactivation by depolarization of Ca²⁺ entry and secretion occluded the blocking effects of combined -conotoxin GVIA (1 µM) and -agatoxin IVA (2 µM), thus suggesting that depolarization caused a selective inactivation of the N- and P/Q-type Ca²⁺ channels.

6. This was strengthened by two additional findings: (i) nifedipine (3 µM), an L-type Ca²⁺ channel blocker, suppressed the fraction of Ca²⁺ entry (24 %) and secretion (27 %) left unblocked by depolarization; (ii) FPL64176 (3 µM), an L-type Ca²⁺ channel 'activator', dramatically enhanced the entry of Ca²⁺ and the secretory response in depolarized cells.

7. In voltage-clamped cells, switching the holding potential from -80 to -40 mV promoted the loss of 80 % of the whole-cell inward Ca²⁺ channel current carried by 10 mM Ba²⁺ (I_Ba). The residual current was blocked by 80 % upon addition of 3 µM nifedipine and dramatically enhanced by 3 µM FPL64176.

8. Thus, it seems that the N- and P/Q-subtypes of calcium channels are more prone to inactivation at depolarizing voltages than the L-subtype. We propose that this different inactivation might occur physiologically during different patterns of action potential firing, triggered by endogenously released acetylcholine under various stressful conditions.

	INTRODUCTION

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A variety of voltage-dependent Ca²⁺ channels exhibiting similar biophysical and pharmacological characteristics as those described in neurones (Olivera et al. 1994) are expressed in bovine chromaffin cells (Albillos et al. 1996b; for a more extensive review see García et al. 1997). The question arises as to why a cell specialized to secrete catecholamines by exocytosis requires more than one Ca²⁺ channel subtype to deliver the Ca²⁺ necessary to activate the secretory machinery. This question is also highly relevant for many brain synapses, since the release of their neurotransmitters is also controlled by more than one Ca²⁺ channel type (Olivera et al. 1994; García et al. 1997).

The L- and non-L-type (N/P/Q) Ca²⁺ channels found in bovine chromaffin cells contribute to an increase in ⁴⁵Ca²⁺ entry (Ballesta et al. 1989; López et al. 1994b; Villarroya et al. 1997), and to a rise in the cytosolic Ca²⁺ concentration, ([Ca²⁺]_i) (Rosario et al. 1989; López et al. 1994a; Lomax et al. 1997) and secretion (López et al. 1994b; Lara et al. 1998). The need to generate different specific secretory responses both qualitatively and quantitatively, under different physiologically or pathologically stressful stimuli, may account for the co-expression of various Ca²⁺ channel subtypes in the same cell. Although different mechanisms may shape this secretory response (see Discussion), one possible means of selecting a given Ca²⁺ entry pathway would be the occlusion of other pathways through the use of voltage-dependent mechanisms.

Electrophysiological patch-clamp experiments have shown that chromaffin cell Ca²⁺ channels undergo both Ca²⁺- and voltage-dependent inactivation. Thus Ba²⁺ carries more than double the current carried by Ca²⁺(I_Ca), and I_Ca inactivates while I_Ba does not (Albillos et al. 1994). These are clear signs of Ca²⁺-dependent Ca²⁺ channel inactivation similar to those seen in the pioneering works of Brehm & Eckert (1978) and Tillotson (1979). Voltage-dependent inactivation of Ca²⁺ channels has also been shown in bovine chromaffin cells (Hans et al. 1990; but see Artalejo et al. 1992), a behaviour reminiscent of dorsal root ganglion neurones (Nowicky et al. 1985). Using other methodologies in intact cells (⁴⁵Ca²⁺ uptake, fluorometric techniques) we have previously shown that the Ca²⁺ channels of bovine chromaffin cells also inactivate in a voltage- and Ca-dependent manner (Artalejo et al. 1987; Fonteríz et al. 1992; Michelena et al. 1993). This inactivation blocks the entry of Ca²⁺ in parallel with a blockade of catecholamine release in K⁺-depolarized bovine (Artalejo et al. 1986; Michelena et al. 1993) and cat (Garrido et al. 1990) chromaffin cells. However, we do not know if L- and N/P/Q-types of Ca²⁺ channels suffer an equal or different voltage-dependent inactivation. Here we present a study aimed at answering these questions through the measurement of whole-cell inward calcium channel currents, ⁴⁵Ca²⁺ entry, changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and catecholamine release (secretion) in bovine adrenal medullary chromaffin cells.

	METHODS

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Bovine adrenal glands were obtained from a local slaughterhouse. Adrenal chromaffin cells were isolated following standard methods (Livett, 1984) with some modifications (Moro et al. 1990). Cells were suspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5 % fetal calf serum, 10 µM cytosine arabinoside, 10 µM fluorodeoxyuridine, 50 i.u. ml^-1 penicillin and 50 µg ml^-1 streptomycin. Cells were kept in a water-saturated incubator at 37°C, in a 5 % CO₂-95 % air atmosphere, and used 1-3 days thereafter.

Measurement of whole-cell currents through calcium channels

For measurements of ionic currents cells were plated on 1 cm diameter glass coverslips at low density (5 × 10⁴ cells per coverslip). Ba²⁺ currents through Ca²⁺ channels were recorded using the whole-cell configuration of the patch-clamp technique (Hamill et al. 1981). Coverslips containing the cells were placed in an experimental chamber mounted on the stage of a Nikon Diaphot inverted microscope. The chamber was continuously perfused with a control Tyrode solution containing (mM): NaCl, 137; MgCl₂, 1; CaCl₂, 2; Hepes-NaOH, 10; tetrodotoxin (TTX), 0·005; pH 7·4. For current recording, 10 mM Ba²⁺ (instead of 2 mM Ca²⁺) was used as a charge carrier. Cells were dialysed with a standard intracellular solution containing (mM): NaCl, 10; CsCl, 100; TEACl, 20; MgATP, 5; EGTA, 14; Hepes-CsOH, 20; pH 7·2.

Whole-cell recordings were made with fire-polished electrodes (resistance 2-5 M when filled with the standard Cs⁺-TEA intracellular solution) mounted on the headstage of a Dagan 8900 patch-clamp amplifier, allowing cancellation of capacitative transients and compensation of series resistance. A Labmaster data acquisition and analysis board and a 386-based microcomputer with pCLAMP software (Axon Instruments) were used to acquire and analyse the data.

Cells were clamped at a holding potential (V_h) of -80 mV. Step depolarizations to 0 mV from this V_h lasted 50 ms and were applied at 0·1 Hz to minimize the run-down of Ca²⁺ currents (Fenwick et al. 1982). Cells with pronounced run-down were discarded. Leak and capacitative currents were subtracted using currents elicited by small hyperpolarizing pulses.

External solutions were exchanged using electronically driven miniature solenoid valves coupled to a multi-barrelled concentration-clamp device, the common outlet of which was placed within 100 µm of the cell from which the patch would be obtained. The flow rate was low (< 1 ml min^-1) and regulated by gravity to achieve complete replacement of the cell surroundings within less than 1 s. Experiments were performed at room temperature (22-24°C).

Experimental design

The experimental approach when measuring ⁴⁵Ca²⁺ entry, [Ca²⁺]_i and catecholamine release was first to expose cells to hyperpolarizing (1·2K⁺/0Ca²⁺) or depolarizing (100K⁺/0Ca²⁺) solution (in which NaCl was iso-osmotically replaced by KCl). Subsequently, the stimulation of Ca²⁺ entry, the [Ca²⁺]_i rise and the increment of catecholamine release were triggered by the application of 5 s pulses of modified depolarizing solution containing 2 mM Ca²⁺ (100K⁺/2Ca²⁺; the Ca²⁺ pulse). These experiments were performed in control cells, or in cells treated with selective modulators (blockers or activator) of L and non-L Ca²⁺ channel subtypes. Because we were dealing with inactivation of metabolically dependent processes (for instance, the exocytotic Ca²⁺-dependent release of catecholamines is ATP dependent; Baker & Knight, 1978), we decided to perform all ⁴⁵Ca²⁺ entry and catecholamine release experiments at a physiological temperature (37°C). Because of technical limitations, the fura-2 measurements of [Ca²⁺] changes were performed at a relatively high room temperature (26-28°C) and the patch-clamp experiments at room temperature (22-24°C).

Measurement of catecholamine release

For secretion experiments, cells (5 × 10⁶) were plated on plastic 6 cm diameter Petri dishes containing 5 ml DMEM. Catecholamine release (secretion) was measured by placing the cells (2-3 days in culture) inside a microchamber and superfusing them with a basal Krebs-Hepes solution of the following composition (mM): NaCl, 140; KCl, 5·9; MgCl₂, 1·2; CaCl₂, 2; Hepes-NaOH, 15; pH 7·4. Cells were subsequently superfused with a nominally Ca²⁺-free Krebs-Hepes solution containing either 100 mM K⁺ (100K⁺/0Ca²⁺ or depolarizing solution) or 1·2 mM K⁺ (1·2K⁺/0Ca²⁺ or hyperpolarizing solution) in which NaCl was replaced iso-osmotically by KCl. In some experiments, Ca²⁺-free solutions containing various concentrations of K⁺ (iso-osmotically replacing NaCl) were also used. Catecholamine release was triggered by intermittently applying brief 5 s pulses of modified depolarizing Krebs-Hepes solution containing 2 mM Ca²⁺. Basal and evoked catecholamine release were monitored continuously on-line with an electrochemical detector (Borges et al. 1986).

Measurement of ⁴⁵Ca²⁺ uptake

For ⁴⁵Ca²⁺ uptake experiments cells were seeded on 96-multiwell plates at a density of 2 × 10⁵ cells per microwell. ⁴⁵Ca²⁺ uptake studies were carried out in cells after 2-3 days in culture. Before the experiment, cells were washed twice with 0·2 ml Krebs-Hepes solution, of the following composition (mM): NaCl, 140; KCl, 5·9; MgCl₂, 1·2; CaCl₂, 1; glucose, 11; Hepes, 10; pH 7·2.

⁴⁵Ca²⁺ uptake into chromaffin cells bathed in hyperpolarizing or depolarizing solutions was studied by applying a 5 s pulse of a solution containing 100 mM K⁺, 2 mM Ca²⁺ and 5 µCi ml^{-1 45}Ca²⁺ (100K⁺/2Ca²⁺/⁴⁵Ca²⁺). At the end of the 5 s pulse period, the test medium was rapidly aspirated and 0·2 ml of cold Ca²⁺-free Krebs-Hepes solution containing 10 mM LaCl₃ was added. Finally, the cells were washed five more times with 0·2 ml of Ca²⁺-free Krebs-Hepes solution containing 10 mM LaCl₃ and 2 mM EGTA at 15 s intervals.

To measure the radioactivity retained, 0·2 ml of 10 % trichloroacetic acid was added to each well and cells were scraped with a plastic pipette tip and transferred to scintillation minivials. Then, 3·5 ml scintillation fluid (Ready Micro, Beckman) were added and the samples counted in a Packard beta counter. Results are expressed as counts per minute (c.p.m.) per 2 × 10⁵ cells or as net Ca²⁺ entry (fmol cell^-1). All the experiments were counted in the same beta counter with an efficiency of 60 %; therefore, the quenching was always the same. For this reason, the results are expressed in counts rather than disintegrations per minute.

Measurement of changes in [Ca²⁺]_i in fura-2-loaded cells

To measure the changes in [Ca²⁺]_i cells were plated on 1 cm diameter glass coverlips at low density (5 × 10⁴ cells per coverslip). Chromaffin cells were loaded with fura-2 by incubating them with fura-2 AM (6 µM) for 45 min at 37°C in the dark in Krebs-Hepes solution (pH 7·4) containing (mM): NaCl, 140; KCl, 5·9; MgCl₂, 1·2; CaCl₂, 2; Na-Hepes, 10; glucose, 11; pH 7·4. The loading incubation was terminated by washing the coverslip containing the attached cells several times with Krebs-Hepes solution. Then, cells were kept at room temperature (26-28°C) for 15-30 min. The fluorescence of fura-2 in single cells was measured with the photomultiplier-based system described by Neher (1989), which produces a spatially averaged measure of the [Ca²⁺]_i. Fura-2 was excited with light alternating between 360 and 390 nm, using a Nikon × 40 fluorite objective lens. Emitted light was transmitted through a 425 nm dichroic mirror and 500-545 nm barrier filter, before being detected by the photomultiplier. [Ca²⁺]_i was calculated from the ratios of the light emitted when the dye was excited by the two alternating excitation wavelengths (Grynkiewicz et al. 1985). Experiments were performed at 26-28°C, using protocols and solutions similar to those described for catecholamine release studies.

Materials and solutions

The following materials were used. Clostridium histolyticum collagenase (Boehringer-Mannheim); bovine serum albumin fraction V (Sigma). DMEM and antibiotics were from Gibco. Nifedipine was from Sigma. FPL64176 was from RBI. TTX was from Calbiochem. -Agatoxin IVA was from The Peptide Institute and -conotoxin GVIA was from Bachem Feinchemikalien.

-Conotoxin GVIA and -agatoxin IVA were dissolved in distilled water and stored frozen in aliquots at 0·1 mM. Nifedipine (10 mM) and FPL64176 (1 mM) were prepared in dimethylsulphoxide and polyethyleneglycol, respectively, and diluted to the required final concentration (3 µM) in Krebs-Hepes solution. At these dilutions, solvents had no effect on the parameters studied.

Statistics

Data are expressed as means ± S.E.M. Statistical differences between means were estimated using Student's t test for paired and non-paired group data; P values smaller than 0·05 were taken as significant.

	RESULTS

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Loss of calcium channel current at depolarizing holding potentials

One direct approach for studying the possible selective voltage inactivation of channel subtypes is the measurement of whole-cell inward calcium channel current using patch-clamp techniques. In the experiment shown in Fig. 1A the cell was initially voltage clamped at -80 mV. Test depolarizing pulses to 0 mV given at 10 s intervals produced peak Ba²⁺ currents (I_Ba) of around 750 pA. Then, the holding potential was switched from -80 to -40 mV; I_Ba started to decline immediately and reached a new steady-state value of around 200 pA after 3-4 min. In 21 cells, this mild depolarization of the cells reduced I_Ba by 75 ± 3 %, from 783 ± 89 pA at -80 mV to 188 ± 254 pA at -40 mV. Nifedipine (3 µM) further depressed this residual current. A return to the original holding potential (-80 mV) favoured the gradual partial recovery of I_Ba to about 500 pA.

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Figure 1. Inactivation by voltage of N- and P/Q- but not L-type calcium channel currents The cell in A was initially voltage clamped at -80 mV, then at -40 mV, and finally back to -80 mV (as indicated by the upper bar). Nifedipine (3 µM) was applied as shown. Depolarizing test pulses to 0 mV were given at 10 s intervals to elicit inward calcium channel currents using 10 mM Ba2+ as charge carrier (IBa); peak current (in pA) versus time (in s) are plotted. Inset, examples of original IBa traces taken at points a and b. B shows the L- and N/P/Q calcium channel current components identified using 3 µM nifedipine or 1 µM -conotoxin GVIA (GVIA) plus 2 µM -agatoxin IVA (Aga) respectively, at a Vh of -80 or -40 mV. Data are means + S.E.M. of the number of cells indicated in parentheses above each column. P < 0·001 when compared with the respective controls (Student's paired t test); **P < 0·01 and ***P < 0·001 when values at Vh -40 mV were compared with those at Vh -80 mV. The cell recorded from in C was voltage clamped at -40 mV and after 5 min a control I-V curve was observed. A second I-V curve was repeated in the presence of 3 µM FPL64176. Insets show original current traces taken at test pulses of -20 mV (top) and -10 mV (bottom) in the absence (Control) and the presence of FPL64176 (FPL). Time calibration for top inset applies also to the bottom inset.

The use of selective calcium channel blockers allowed the determination of the contribution of each channel type (L or N/P/Q) to the whole-cell current at -80 mV holding potential, as well as the estimation of the channel subtype lost at -40 mV holding potential (Fig. 1B). Thus, of the 700 pA I_Ba found in control conditions at -80 mV, about 15 % was sensitive to 3 µM nifedipine (L channels) and 80 % was sensitive to 1 µM -conotoxin GVIA (N channels) plus 2 µM -agatoxin IVA (P/Q channels). Since N/P/Q channels accounted for nearly 80 % of the current, and at a holding potential of -40 mV about 70 % of the current was lost, it seems obvious that the inactivation of the N and P/Q channels must be related to the current lost at the depolarized holding potential. The residual current at -40 mV was highly sensitive to nifedipine (blockade of 57 ± 5 %, n = 7; P < 0·001 compared with blockade at -80 mV) but also to the -toxins, suggesting that some current associated with N/P/Q channels (10 %) remained at the depolarized potential.

The residual current at -40 mV was also highly sensitive to 3 µM FPL64176, as shown in Fig. 1C. Here the cell was voltage clamped at -40 mV and after the current stabilized at about 120 pA an I-V curve was obtained (Control). In the presence of FPL64176 current activation was drastically slowed, the magnitude of the current (particularly at -20 and -10 mV test potentials) was increased and a pronounced delay of I_Ba deactivation (see original current traces in the inset to Fig. 1C) was induced. As a consequence of this, the I-V curve obtained in the presence of FPL64176 was shifted to the left.

Ca²⁺ entry into hyperpolarized and depolarized chromaffin cells induced by the Ca²⁺ pulse

Ca²⁺ entry was studied by monitoring ⁴⁵Ca²⁺ uptake and the increments of [Ca²⁺]_i. ⁴⁵Ca²⁺ uptake was studied following the protocols shown at top of Fig. 2. Hyperpolarized chromaffin cells stimulated with the Ca²⁺ pulse solution (containing traces of ⁴⁵Ca²⁺) incorporated 1307 ± 44 c.p.m. well^-1, while depolarized cells retained 578 ± 25 c.p.m., and resting cells retained 345 ± 15 c.p.m. (basal ⁴⁵Ca²⁺ uptake). To determine the uptake of 'cold Ca²⁺' (⁴⁰Ca²⁺, expressed in fmol cell^-1) from ⁴⁵Ca²⁺ uptake (in c.p.m.) we estimated the specific activity of the ⁴⁵Ca²⁺/⁴⁰Ca²⁺ present during the Ca²⁺ pulse (2 mM ⁴⁰Ca²⁺) by counting a 10 µl aliquot of the Krebs-Hepes solution, and subtracted the basal ⁴⁵Ca²⁺ taken up by cells (hyperpolarized cells exposed for 5 s to a 1·2K⁺/2Ca²⁺/⁴⁵Ca²⁺ solution) (Basal bars in Fig. 2). Net Ca²⁺ uptake was calculated by subtracting the basal from the evoked Ca²⁺ uptake; in hyperpolarized cells it amounted to 0·65 ± 0·02 fmol cell^-1 while in depolarized cells the net entry of Ca²⁺ was markedly reduced to 0·16 ± 0·015 fmol cell^-1. Assuming the chromaffin cell to be a sphere of 20 µm diameter with a uniform intracellular Ca²⁺ distribution, the cytosolic Ca²⁺ concentration reached in hyperpolarized cells was 31·5 µM in 1 s; in depolarized cells the calculated [Ca²⁺]_i reached 7·8 µM in 1 s. If a cytosolic water space of 50 % is assumed (Artalejo et al. 1987), the [Ca²⁺]_i reached in 1 s should be 63 µM in hyperpolarized cells and 15·6 µM in depolarized cells.

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Figure 2. Ca2+ entry into depolarized cells was dramatically reduced with respect to hyperpolarized cells To measure the basal 45Ca2+ uptake, cells were bathed in 1·2 mM K+ during the preincubation period (10 min) and in 1·2K+/2Ca2+/45Ca2+ for 5 s. Hyperpolarized cells were bathed in 1·2K+/0Ca2+ and depolarized cells in 100K+/0Ca2+ for 10 min; the subsequent 45Ca2+ uptake was stimulated with a 5 s pulse of 100K+/2Ca2+/45Ca2+ (see protocols on top of bars). Data are means ± S.E.M. of the number of wells shown in parentheses, from 2 different cell cultures. **P < 0·01 compared with basal value; P < 0·001 compared with depolarized value.

The increments in [Ca²⁺]_i in fura-2-loaded cells paralleled those of ⁴⁵Ca²⁺ entry. The Ca²⁺ pulses produced [Ca²⁺]_i peaks of 1·2 ± 0·1 µM in hyperpolarized cells (n = 65 Ca²⁺ pulses) and 0·11 ± 0·008 µM in depolarized cells (n = 72 Ca²⁺ pulses) (P < 0·001).

Catecholamine release from depolarized and hyperpolarized cells, triggered by sequential Ca²⁺ pulses: correlation with the increments in [Ca²⁺]_i

If depolarization inactivates Ca²⁺ entry, it follows that depolarization should also provoke the inactivation of the Ca²⁺-dependent catecholamine release response evoked by Ca²⁺ pulses. This is shown in Fig. 3. In Fig. 3A, cells were initially superfused with a hyperpolarizing solution and then with a depolarizing solution. The application of Ca²⁺ pulses induced sharp secretory spikes that were very reproducible while cells were being hyperpolarized; the spikes reached values close to 1000 nA. On switching to a depolarizing solution, the Ca²⁺ pulses produced secretory spikes more than 10-fold smaller in magnitude than those seen in hyperpolarized cells. Figure 3B shows a similar experiment but here the cells were first superfused with the depolarizing solution and then with the hyperpolarizing solution. The initial small secretory spikes were rapidly enhanced upon switching from 1·2K⁺/0Ca²⁺ to 100K⁺/0Ca²⁺ solution. Figure 3C shows the averaged values of several experiments performed in separated cells that were either hyperpolarized (n = 4) or depolarized (n = 5) for the entire experiment; nine Ca²⁺ pulses were applied to each individual cell batch. Secretion spikes increased initially to reach stable peaks at around 900 nA; in depolarized cells the peaks had an initial magnitude of about 180 nA and then, declined to 103 nA by the ninth peak. Figure 3C also shows the averaged [Ca²⁺]_i spikes generated by Ca²⁺ pulses sequentially applied to fura-2-loaded cells, under depolarizing or hyperpolarizing conditions. Note that the magnitudes of the Ca spikes paralleled those of the secretion peaks obtained in hyperpolarized and depolarized cells. Data for [Ca²⁺]_i are means ± S.E.M. of 7-9 cells.

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Figure 3. The magnitude of the catecholamine secretory spikes produced by sequential Ca2+ pulses in superfused cells was dramatically reduced in depolarized compared with hyperpolarized cells In A, chromaffin cells were initially superfused with 1·2K+/0Ca2+ and subsequently with 100K+/0Ca2+; in B, the order of the solutions was reversed, as shown by the horizontal bars at the bottom of each figure. Ca2+ pulses (indicated by ) were applied at 2 min intervals. C shows averaged data of catecholamine secretory peaks (left ordinate) and [Ca2+]i peaks (right ordinate), elicited by sequential Ca2+ pulses (abscissa) given to cells superfused continuously with hyperpolarizing or depolarizing solutions, following protocols similar to those used for secretion, but in fura-2-loaded single cells. Data are means ± S.E.M. of 4-5 experiments of secretion and 7-9 experiments of Ca2+i. Data were obtained from at least 3 different cell cultures.

Ca²⁺ entry evoked by Ca²⁺ pulses in hyperpolarized or depolarized cells exposed to selective Ca²⁺ channel modulators

The next step was to clarify whether the inactivation of Ca²⁺ entry occurred preferentially through a specific Ca²⁺ channel subtype, or indistinctly at all Ca²⁺ channel subtypes. To perform these experiments, the channels were pharmacologically separated into L- and non-L (N/P/Q)-subtypes (Olivera et al. 1994; García, et al. 1997). N/P/Q channels were blocked by preincubation of the cells for 45 min with 1 µM -conotoxin GVIA (N channel blockade) plus 2 µM -agatoxin IVA (P/Q channel blockade). To block the L-type Ca²⁺ channels, 3 µM nifedipine was continuously used throughout the experiment; to prolong the opening time of L channels, cells were continuously exposed to 3 µM FPL64176 (McKechnie et al. 1989; Rampe & Dage, 1992).

In hyperpolarized cells, the blockade of N/P/Q Ca²⁺ channels with combined -conotoxin GVIA and -agatoxin IVA reduced the net ⁴⁵Ca²⁺ entry by 66 % with respect to untreated control cells. In depolarized cells, the control ⁴⁵Ca²⁺ entry was drastically reduced from 947 to 129 c.p.m.; preincubation with the toxins did not further reduce the ⁴⁵Ca²⁺ uptake (Fig. 4A).

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Figure 4. Ca2+ entry into hyperpolarized and depolarized cells treated with specific Ca2+ channel blockers and activator Hyperpolarized cells were incubated for 10 min in 1·2K+/0Ca2+ solution and depolarized cells in 100K+/0Ca2+solution. -Conotoxin GVIA (GVIA, 1 µM) and -agatoxin IVA (Aga, 2 µM) were present for 45 min, followed by a 20 min washout period, before challenging them with the Ca2+ pulse (A). Nifedipine (Nif, 3 µM) and FPL64176 (FPL, 3 µM) were present for 10 min before applying the Ca2+ pulse (B). The conditions were similar to those used for secretion experiments (Fig. 3). Net 45Ca2+ entry was calculated by subtracting basal 45Ca2+ entry from the total 45Ca2+ retained by the cells. Data are means + S.E.M. of the number of wells shown in parentheses, from at least 2 different cell cultures. ***P < 0·001 compared with their respective controls.

The effects of L-type channel modulators are shown in Fig. 4B. In hyperpolarized cells the control ⁴⁵Ca²⁺ uptake following the Ca²⁺ pulse amounted to 812 c.p.m. Nifedipine reduced ⁴⁵Ca²⁺ uptake by 30 % and FPL64176 by 20 %. It is puzzling that the L-type Ca²⁺ channel activator did not act in hyperpolarized cells to enhance the opening of such channels. There might be a voltage-dependent binding of the compound to its receptor. In depolarized cells, however, FPL64176 dramatically enhanced the uptake of ⁴⁵Ca²⁺, which had been previously been reduced by 55 % by exposing the cells to 100K⁺/0Ca²⁺ solution for 10 min. Nifedipine blocked almost to completion the ⁴⁵Ca²⁺ uptake signal generated by the Ca²⁺ pulse in cells previously incubated in 100K⁺/0Ca²⁺ solution.

Catecholamine release from hyperpolarized and depolarized chromaffin cells treated with selective Ca²⁺ channel blockers or activator

The cells that were continuously superfused with a hyperpolarizing solution responded to the Ca²⁺ pulse with catecholamine secretory spikes of 748 ± 31 nA (Fig. 5A). If the cells were pretreated with combined -conotoxin GVIA plus -agatoxin IVA to block irreversibly N/P/Q channels (Albillos et al. 1996b; Gandía et al. 1997), the secretory response was reduced by 30 %. Nifedipine also caused a decrease of about 30 %; FPL64176 caused no significant changes in secretion. The poorer blocking effects of nifedipine under these hyperpolarizing conditions might be due to the voltage dependence of dihydropyridine effects in chromaffin cells (López et al. 1989).

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Figure 5. Catecholamine release responses in hyperpolarized (A) and depolarized (B) chromaffin cells, treated with selective blockers or an activator of their Ca2+ channel subtypes 'Control' cells were superfused continuously with 1·2K+/0Ca2+ solution (hyperpolarized) or 100K+/0Ca2+ solution (depolarized) and stimulated to secrete with 5 s pulses of 100K+/2Ca2+ solution given at 2 min intervals. Aga + GVIA-treated cells were preincubated for 45 min at 37 °C in 0·5 ml Krebs-Hepes solution containing 1 µM -conotoxin GVIA and 2 µM -agatoxin IVA, and then continuously superfused with hyperpolarizing or depolarizing solutions in the absence of toxins. Nife- or FPL-treated cells were superfused with hyperpolarizing or depolarizing solutions containing 3 µM nifedipine or 3 µM FPL64176. All cells were stimulated 5 times with 5 s pulses of 100K+/2Ca2+ solution. Results are means + S.E.M. of individual secretion spikes (in nA pulse-1) (ordinates), taken from at least 3 different cell cultures; values in parentheses indicate number of spikes for each experimental condition. **P < 0·05 and ***P < 0·01 compared with control.

As expected, depolarized cells responded to Ca²⁺ pulses with secretory peaks substantially smaller than those obtained from hyperpolarized cells; the size of averaged peaks amounted to 153 ± 15 nA (n = 60 pulses). Pretreatment of the cells with -conotoxin GVIA plus -agatoxin IVA did not reduce further the release of catecholamines (198 ± 36 nA; n = 20 pulses). However, nifedipine almost completely suppressed the secretory response (33 ± 3 nA, n = 25 pulses). A striking potentiation of secretion was seen with FPL64176 that increased the secretory signal to 422 ± 65 nA (n = 31 pulses) (Fig. 5B).

Catecholamine secretory spikes induced by Ca²⁺ pulsing of cells superfused with increasing K⁺ concentrations in nominally Ca²⁺-free solution

These experiments were designed to test whether the inactivation of secretion spikes was gradual, depending on the [K⁺] of the nominally Ca²⁺-free solution superfusing the cells. Figure 6A shows that between the two extremes of 1·2K⁺/0Ca²⁺ (the hyperpolarizing solution referred to above) and 100K⁺/0Ca²⁺ (the depolarizing solution referred to above), there was a graded inactivation of secretion as the [K⁺] increased. However, there was a brisk jump in the inactivation between the 17·7K⁺/0Ca²⁺ and 35K⁺/0Ca²⁺ solutions. This jump is more clearly shown in Fig. 6B, were secretion peaks obtained with individual Ca²⁺ pulses are plotted as a function of the [K⁺]. Note that the size of secretion peaks decreased from 486 ± 12 nA in 17·7K⁺/0Ca²⁺ to 192 ± 12 nA in 35K⁺/0Ca²⁺. Higher [K⁺] did not cause much more inactivation of secretion.

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Figure 6. Voltage-dependent inactivation of catecholamine secretion peak responses induced by Ca2+ pulses, and its reversal by FPL64176 In the experiments shown in A, chromaffin cells were continuously superfused with nominally Ca2+-free solutions containing increasing concentrations of K+ (1·2, 5·9, 17·7, 35, 59, 70 and 100 mM, KCl iso-osmotically replacing NaCl). Then, 11 pulses of 100K+/2Ca2+ 5 s duration were applied at 2 min intervals. A separate batch of cells was used for each [K+]. The net catecholamine release (evoked minus basal secretion) is expressed as nA pulse-1 (ordinate), and the number of pulses is shown on the abscissa. Data are means ± S.E.M. of the number of cell batches used, given in parentheses to the right of each [K+]. B shows a plot of the magnitude of the peaks of secretion induced by individual Ca2+ pulses, as a function of the [K+] that was superfusing the cells in nominally Ca2+-free solution between the Ca2+ pulses, in the absence (Control) and presence of 3 µM FPL64176 (FPL). Data are means ± S.E.M. of the number of Ca2+ pulses shown in parentheses at each point. *P < 0·05 and ***P < 0·001 compared with the respective [K+] in control cells.

At the end of the experiments shown in Fig. 6A, FPL64176 was introduced and some additional Ca²⁺ pulses were given to cells being superfused with 0Ca²⁺ solutions containing various [K⁺]. The secretory spikes thus obtained were averaged and plotted in Fig. 6B, as a function of the [K⁺]. At all [K⁺] (except 5·9 mM), the magnitude of the responses were significantly higher than those obtained in the absence of FPL64176 (control cells in the figure). Thus, FPL64176 drastically reduced the inactivation of secretion at all [K⁺] tested; obviously, the higher the [K⁺] the greater the inactivation and the larger the effects of the compound.

Inactivation of secretion spikes induced by Ca²⁺ pulses, as a function of the length of depolarization

In this experiment we explored how fast the inactivation of secretory peak responses developed in cells superfused with a depolarizing solution (100K⁺/0Ca²⁺). Figure 7A shows a prototype experiment. Cells initially superfused with a hyperpolarizing solution (1·2K⁺/0Ca²⁺) produced secretory spikes of around 800 nA upon their challenging with 5 s 100K⁺/2Ca²⁺ pulses. A 2 s period of superfusion with 100K⁺/0Ca²⁺ preceding the Ca²⁺ pulse did not reduce the size of the spike. The increase of the exposure to this depolarizing solution produced a gradual decline of the secretory spike, which was reduced to 235 nA after 2 min. Averaged results of the time-dependent inactivation of secretion appear in Fig. 7B. Depolarization caused inactivation of the response with a half-time for inactivation of about 1 min.

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Figure 7. Time-dependent inactivation of the secretory peak responses produced by Ca2+ pulses applied to chromaffin cells superfused with a depolarizing solution In A cells were initially superfused with 1·2K+/0Ca2+ solution; after 3 initial 5 s pulses with 100K+/2Ca2+ solution applied at 2 min intervals (3 first secretion spikes) cells were superfused with a depolarizing solution (100K+/0Ca2+) for increasing lengths of time, immediately before the application of the subsequent Ca2+ pulse. The number above each spike represents the time in seconds of cell exposure to the depolarizing solution, before the application of the Ca2+ pulse. B shows averaged experiments performed with the protocol shown in A. Data are means ± S.E.M. of 6 experiments.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

The experiments performed in this study show that bovine chromaffin cells exposed to a strong depolarizing K⁺-enriched solution, in the absence of extracellular Ca²⁺ (nominally 0Ca²⁺), caused the inactivation of the catecholamine secretory responses induced by the subsequent application of brief Ca²⁺ pulses. This inactivation is time and voltage dependent since increasing [K⁺] and length of depolarizations, caused increased levels of inactivation (Figs 6 and 7). The reduction in secretion is surely due to a drastic decrease in extracellular Ca²⁺ entry and the subsequent reduction of [Ca²⁺]_i during the Ca²⁺ pulse, as proven by the experiments of Figs 2 and 3.

Because the bovine chromaffin cell expresses L-type as well as non-L-type Ca²⁺ channels (see Introduction for references), the question arises as to whether the inactivation of Ca²⁺ entry is a consequence of the equal inactivation of all subtypes of calcium channels, or the selective inactivation of specific calcium channel subtypes. Two experimental facts support the last option: (i) depolarization largely occluded the blocking effects of -agatoxin IVA plus -conotoxin GVIA on Ca²⁺ entry and secretion; (ii) nifedipine suppressed and FPL64176 potentiated the fraction of secretion that remained unblocked in depolarized cells. Therefore, it seems appropriate to conclude that N/P/Q-type Ca²⁺ channels (targeted by the toxins) are selectively inactivated in depolarized cells, while L-type Ca²⁺ channels remain functional; this is supported by the restoration of Ca²⁺ entry and secretion in depolarized cells exposed to FPL64176, a molecule that potently increases the mean open time of L-type Ca²⁺ channels in muscle cells (McKechnie et al. 1989; Rampe & Dage, 1992).

The selective voltage inactivation of N/P/Q but not L channels was demonstrated through the direct measurements of whole-cell inward currents flowing through calcium channels in voltage clamped cells (Fig. 1). Switching the holding potential from -80 to -40 mV caused a loss of I_Ba of 75 % in 3-4 min (but see Artalejo et al. 1992 who found no current loss at depolarizing holding potentials). This degree of depolarization (40 mV) is achieved by raising the K⁺ concentration of the extracellular solution by about 30-50 mM in rat (Ishikawa & Kanno, 1978), mouse (Nassar-Gentina et al. 1988) and bovine chromaffin cells (González-Garcia et al. 1993). This establishes a good correlation between the amount of current lost at -40 mV in voltage-clamped cells (75 %) and the fraction of the secretion response lost in cells depolarized with 35-59 mM K⁺ (about 80 %, Fig. 6). The fraction of I_Ba lost accounted mostly for N/P/Q channel inactivation, since the current remaining at -40 mV holding potential was still highly sensitive to nifedipine and to FPL64176. This also correlates well with the results obtained from K⁺ depolarization of cells, measuring ⁴⁵Ca²⁺ entry and catecholamine release.

The regulation of exocytosis in chromaffin cells by Ca²⁺ ions entering the cell through different Ca²⁺ channel pathways might be exerted via various mechanisms. One possible explanation for this would be the physical segregation of given calcium channel classes to specific exocytotic sites. This hypothesis is not supported by the observation that the exocytotic electrochemical peaks measured with carbon fibre electrodes in single bovine chromaffin cells are delayed; this favours the uneven distribution of calcium channels along the entire secretory surface (Chow et al. 1992). However, it may very well be that when chromaffin cells are cultured for several days the more discrete location of calcium channels nearby specific exocytotic sites is lost, as proven by recent experiments performed in single chromaffin cells in situ in mouse adrenal slices; measurement of changes in capacitance induced by short depolarizing stimuli proves that in situ chromaffin cells have a much faster component of exocytosis which indicates that the calcium channels are in much closer proximity to the exocytotic machinery (Moser & Neher, 1997). This view is reinforced by recent experiments showing different blockade of secretion at low and high Ca²⁺ gradients, in bovine chromaffin cells whose L or P/Q calcium channels have been occluded using selective blockers; these experiments suggest that P/Q as well as L channels do provide the Ca²⁺ necessary to trigger secretion (López et al. 1994b) but P/Q channels are located closer to secretory sites than L channels (Lara et al. 1998).

An additional feature of the mechanisms involved in the control by Ca²⁺ of exocytosis is the regulation by neurotransmitters of calcium channels. The facilitation of calcium channel currents by depolarizing prepulses in voltage-clamped bovine chromaffin cells, first observed in Erwin Neher's laboratory (Fenwick et al. 1982), seems to be due to tonic inhibition of calcium channels induced by an autocrine/paracrine modulation by materials (i.e. ATP, opiates) coreleased with the catecholamines. The fact that N/P/Q channels are modulated in a voltage-dependent manner and L-type channels in a voltage-independent manner, introduces a distinction between the two groups of channels that could be relevant to their mode of providing Ca²⁺ ions to the secretory machinery under different circumstances. This idea, expressed in a previous communication (Albillos et al. 1996a) is now strengthened by the surprising finding in the present study that voltage changes of the membrane potential clearly distinguishes between L- and non-L-types of Ca²⁺ channels as far as their capacity to deliver Ca²⁺ ions to the secretory machinery, and to trigger the catecholamine release process is concerned.

The first experiments from our laboratory using calcium channel blockers to study the relationship between calcium channels and secretion demonstrated that nifedipine efficiently blocked and Bay K 8644 dramatically potentiated the release of catecholamines triggered by K⁺ depolarization (García et al. 1984) or by Ca²⁺ pulses (Montiel et al. 1984) in perfused cat adrenal glands. This gave rise initially to the wrong idea that only L-type channels were involved in the control of the Ca²⁺ delivery to the secretory machinery of chromaffin cells. This idea had been previously suggested in primary cultures of bovine chromaffin cells, where nitrendipine was seen to block completely the K⁺-evoked release of catecholamines (Ceña et al. 1983). In those earlier studies, and in several other studies from our and other laboratories (see García et al. 1997 for references), secretion was triggered by exposure of the cells to high [K⁺] during long periods of time (1-10 min). As shown in the present study, those conditions would provoke the inactivation of N/P/Q channels, thus masking their possible participation in the triggering of secretion. This inactivation occurs at K⁺ concentrations as low as 17·7 mM and was very pronounced at 35 mM (Fig. 6A). Thus, in the light of these results it is understandable that those earlier experiments ignored the N/P/Q-type Ca²⁺ channels, simply because the experimental conditions used masked their participation in the control of exocytosis.

Another point relates to the relative contribution of L- and P/Q-type Ca²⁺ channels to the K⁺ secretory response. Using -conotoxin MVIIC (López et al. 1994b) or -agatoxin IVA (Lara et al. 1998) we previously suggested that P/Q channels participate in the control of secretion in a proportion similar to L channels. However, in the experiments of Fig. 5B the blockade of secretion by -agatoxin IVA plus -conotoxin GVIA amounted to only 30 %, while ⁴⁵Ca²⁺ entry was inhibited by 70 %. This may be due to differences in protocol (a hyperpolarizing solution containing 1·2 mM K⁺ was used here), or to a partial washout of -agatoxin IVA from the fast-superfused chromaffin cells used here to study secretion. It may also be that under the present experimental conditions L-type channels are more efficient at evoking secretion than N-type and P/Q-type channels.

The final point concerns the question of why chromaffin cells require the expression of voltage-inactivating and voltage-resistant calcium channels to control the delivery of Ca²⁺ to its secretory machinery. L channels remaining functional in spite of sustained cell depolarization might be necessary to support a minimum rate of secretion under durable stress conditions. Ca²⁺ entering through these channels can sustain the release of catecholamines at about 20 % of maximum. On the other hand, N/P/Q channels might have an important physiological role in triggering an explossive catecholamine release during the initial stages of a stressful conflict; later on, these channels might inactivate to switch the command of the control of lower rates of exocytosis to L channels.

In addition to these different roles of Ca²⁺ channel subtypes in controlling exocytosis, certain calcium channels might become specialized in the control of cell functions not immediately related to exocytosis. For instance, L-type calcium channel blockers prevent the changes in gene expression induced by membrane depolarization in cortical (Murphy et al. 1991) and hippocampal neurones (Bading et al. 1993); while N- or P/Q-types of Ca²⁺ channels are involved in the control of transmitter release in nerve terminals (García et al. 1997).

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Acknowledgements

Supported by grants to AGG from Dirección General de Investigación Científica y Técnica (DGICYT No. PB94-0150) and Acción Coordinada de la Comunidad Autónoma de Madrid (CAM No. 07/050/96 and 08.9/0001/1997), Spain. M. F. C.-A. is a fellow of Formaciòn de Personal Investigador, Ministerio de Educación y Cultura, Spain and I. M. is a fellow of Fundación Teófilo Hernando, Spain. We thank Mrs M. C. Molinos for the typing of this manuscript.

Corresponding author

A. G. García: Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain.

Author's present address

R. B. Lomax: Department of Physiology, University of Liverpool, The Physiological Laboratory, Crown Street, Liverpool L69 3BX, UK.

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